Type 1 diabetes mellitus (T1DM) is an autoimmune disease resulting from the T cell mediated destruction of insulin-producing beta cells located in the pancreas. Current treatment, which includes insulin replacement by injection, frequent blood glucose monitoring, and dietary/exercise discipline, can prevent death from hormonal insufficiency, but is not curative and does not completely prevent the long-term complications including nerve damage, and vascular damage to both large and small blood vessels. In previous few years, we developed a transgenic mouse to express the T cell costimulatory receptor CD80 on its pancreatic insulin producing beta cells (under the control of the rat insulin promoter abbreviated RIP) and demonstrated the mouse?s extraordinary sensitivity to autoantigen induced immune mediated beta cell destruction, and thus to diabetes. We refer to the diabetes induced in these RIP-CD80 mice as experimental autoimmune diabetes (EAD). Using this EAD model, we?ve found that relatively weak anti-beta cell autoimmune responses can cause chronic progressive and eventually complete beta cell destruction resulting in symptomatic and irreversible disease. The slow but inexorable process is highly reminiscent of the beta cell destruction leading to clinical type 1 diabetes mellitus (T1DM) in man, typically months to years of anti-beta cell immune activity precedes sufficient beta cell killing for the blood sugars to rise. Most other autoimmune diabetes studies involve the non-obese diabetic (NOD) mouse, which develops spontaneous diabetes, or virus-induced diabetes models. Only the RIP-CD80 transgenic mouse diabetes model is characterized by an experimental genetic susceptibility trait (in the EAD model the trait is the CD80-transgene) rendering the mouse susceptible to autoantigen-specific T lymphocyte sensitization as is thought to be present in T1DM patients. We have reported that immunizing with either experimentally-introduced autoantigen (e.g. pancreatic beta cell-expressed viral glycoprotein) or endogenous beta cell autoantigen (e.g. insulin) could lead to diabetes in RIP-CD80 mice. Further, most studies now support that the normal individual?s T cell repertoire contains potentially autoreactive but quiescent T cells. We concluded that pancreatic beta cell likely contain many autoantigens and that effective control mechanisms must exist to prevent autoimmune responses in healthy individuals. Current immunological dogma suggests that naive CD8 cytotoxic T cell (CTL) precursors respond to strong antigen stimulation in a characteristic fashion by: (i) proliferating and releasing inflammatory cytokines, (ii) differentiating into CTLs and, (iii) down modulating certain surface interaction molecules to allow the CTLs to leave secondary lymphoid organs while increasing other receptors, such as integrins, to promote entry into peripheral tissues, and (iv) changing chemokine signals necessary to facilitate the relocation of activated CTL from lymph nodes (LN) to inflamed peripheral tissues. The EAD model has allowed us to study the response of beta cell-specific CD8 T cells to cognate antigen presented by either professional antigen-presenting cells (APC) like mature dendritic cells [DC]), or by non-professional (np) APCs like fibroblast-like cell lines (FCL). While DC-stimulated T cells produced the expected effector CTL phenotype described above, FCL-activated T cells were quite different. Relative to DC activated CTLs, the FCL-activated CTLs proliferated less, released equivalent proinflammatory cytokines, and surprisingly displayed increased cytolytic function. Moreover, FCL-stimulated T cells largely failed to switch their homing receptors predicting poor migration from the lymph nodes to the periphery. Most strikingly, however, FCL-stimulated but not DC-activated CTL expressed many of the features associated with memory CTLs; both multiple memory cell surface marker expression and predominant homing into secondary lymphoid organs upon adoptive transfer into naive mice. Importantly, while every CTL response gives rise to a small population of long-lived memory CTL, the mechanism that decides the fate of an individual CTL to become a memory cell remains largely unknown. Finally, the FCL-stimulated CTL induced diabetes by a slow, chronic process, suggesting that these central memory CTL might also be involved in driving human T1DM. During this past year we have also established a sensitive and robust in vivo test system to detect putative stem cells for their ability to differentiate into beta cells. Our in vivo mouse EAD model system is designed to test if stem cells can efficiently generate and maintain beta cells during an ongoing autoimmune response. Specific features of the model include: (i) normal host metabolism is maintained for an extended time followed by (ii) pancreatic islet function gradually deteriorates creating a need for new beta cells, (iii) intra-islet inflammation which may provide critical cues for regulated stem cell migration to the pancreas and differentiation into beta cells, and (iv) newly developed, stem cell-derived beta cells are resistant to autoantigen-specific T cell mediated attack. Using this system, we tested crude preparations of hematopoetic stem cells (HSC) reported by some to contain cells capable of transdifferentiating into pancreatic beta cells. We found that the HSC had no functionally relevant capacity to form beta cells in vivo. Using the model, we thoroughly evaluated and experimentally ruled out several critical questions such as whether the failure to generate insulin producing beta cells in vivo might have been due to the inability of newly developed beta cells to withstand the toxicity of an inflammatory environment typically associated with immune-mediated pancreatic islet destruction. Data we?ve generated strongly support the claim that newly generated beta cells from the bone marrow donor origin would be protected from immune mediated attack, by confirming the selective survival of donor-derived pancreatic islets (stem-cell genotype) over host-type islets during rejection of mixed islet transplants. In this past year, we have also developed assays to detect anti-GAD and anti-IA2 autoantibody titers relevant for our T1DM clinical trials. The lab participated in the international Diabetes AutoAntibody Standardization Program (DASP) workshop to validate the laboratory's assays.